Abstract. mAbs specific for titin or nebulin were characterized by immunoblotting and fluorescence microscopy. Immunoelectron microscopy on relaxed chicken breast muscle revealed unique transverse striping patterns. Each of the 10 distinct titin antibodies provided a pair of delicate decoration lines per sarcomere. The position of these pairs was centrally symmetric to the M line and was antibody dependent. The results provided a linear epitope map, which starts at the Z line (antibody T20), covers five distinct positions along the I band (T21, T12, T4, T1, Tll), the A-I junction (T3), and three distinct positions within the A band (T10, T22, T23). The epitope of T23 locates 0.2 txm before the M line. In immunoblots, the two antibodies decorating at or just before the Z line (T20, T21) specifically recognized the insoluble titin TI component but did not recognize TII, a proteolytic derivative. All other titin antibodies recognized TI and TII. Thus titin molecules appear as polar structures lacking over large regions repetitive epitopes. One physical end seems related to Z line anchorage, while the other may bind close to the M line. Titin epitopes influenced by the contractional state of the sarcomere locate between the N1 line and the A-I junction (T4, T1, Tll). We discuss the results in relation to titin molecules having half-sarcomere lengths. The three nebulin antibodies so far characterized again give rise to distinct pairs of stripes. These locate close to the N2 line.T HE pioneering work of the laboratories of Maruyama and Wang has suggested that sarcomeric structure relies not only on the well-known thick and thin filaments with their various associated proteins but also involves an elastic component (for recent reviews see 19,35). While the nature of the elastic filaments is still poorly understood, it seems clear that they are built from very high molecular weight polypeptides (20,22,38). One major component of the system is titin. Its polypeptide molecular weight is variously given as 1 or 2.8 million (21,31,32,39). Titin is usually present as a doublet on low porosity gels and only the second species TII can be purified under native conditions (10,11,31,40). TII is thought to arise by limited proteolysis from the nonextractable TI species (19,35). Although electron micrographs of purified TII indicate morphological heterogeneity, they clearly document very thin and rather long threads. While the exact biophysical properties are still in dispute, length measurements range from ~0.6 to 1.2 Ixm (21,31,40). In agreement with the proposal that it plays a pivotal role in sarcomere integrity, titin is found both in skeletal and cardiac muscle, although possibly not as identical molecules (7,9,17). Much less is known about nebulin from skeletal muscle. Its molecular weight is ~0. 5-0.8 million (21, 35, 38). As it has not been isolated in native form, its function and shape are not known. In addition, there are reports that nebulin is not expressed in cardiac muscle (9,17).There is distinct disagreement about...
